Membrane bound reporter molecules and their use in cell sorting

ABSTRACT

The present invention relates to nucleic acid molecules comprising a nucleic acid sequence encoding a membrane-bound biotin mimetic peptide (BMP) or biotin acceptor peptide (BAP). The invention also relates to a method for selection of high producer cells secreting a protein of interest.

TECHNICAL FIELD

The present invention relates to nucleic acid molecules comprising anucleic acid sequence encoding a membrane-bound biotin mimetic peptide(BMP) or biotin acceptor peptide (BAP). The invention also relates to amethod for selection of high producer cells secreting a protein ofinterest.

BACKGROUND

One of the major challenges in development of producer cells formanufacturing of recombinant proteins is the selection of high producercells among a very large cell population, with different productivitylevels, generated in the course of clone development. The selection ofthe high producers is usually intensive and time consuming. The numberof cells that can be screened manually is limited and so is theprobability to isolate the clones of the required productivity level.

Traditional methods have been employed for screening for high producercells. One of the common manual single cell cloning techniques is the“limiting dilution” procedure. Manual picking and selection of clones bythis procedure is tedious, labour intensive and a time consuming task.Even if performed by robotic systems, the number of clones that can bescreened is limited [1-3]. In addition, statistical analyses indicatethat repeated cloning cycles are required to ensure clonality at anacceptable level. [4, 5]. Therefore, more efficient methods of cell lineselection and cloning are required.

Flow cytometry [6] has been applied for selection of cells labeled withfluorescent reporters. Some of the flow cytometry machines are able tosort thousands of cells within a few seconds and select rare cells withvery low frequencies (as low as 10⁻⁶) in the entire population [7].Another advantage of the flow cytometry is its ability, in some of themachines, to seed single cells (clone the cells) in separate wells inmicrotiter plates [6, 8].

However, cell sorting by flow cytometers requires a fluorescent signal.Since the protein product of interest is not fluorescent in most of thecases, a fluorogenic reporter gene product, whose expression is tightlylinked to that of the gene of interest (GOI), is required.

The reporter gene product may be expressed in the cell cytoplasm or canbe directed to the cell surface [8, 9].

The reporter gene can be naturally fluorescent such as GFP [10].Alternatively, it can be specifically stained with a fluorescence markerthat penetrates the cells such as fluorescent methotrexate (F-MTX) [11]or stained with a fluorescent marker that binds to it on the cellsurface [8, 9].

An interesting case was reported for antibodies produced in cellculture. It has been observed that the secreted antibody itself may beused as a reporter and can be detected on the membrane of hybridomacells [12, 13] and CHO cells [14]. Staining with fluorescently labeledanti-antibodies was used to detect the antibody on cell surface.

A further known method for isolation of specific desired cells is basedon magnetic beads. Magnetic beads coated with a protein that bindsspecifically to a target protein on the cells' surface was previouslyreported [9].

Other methods based on direct detection of the secreted POI were alsopublished. These methods include gel microdrop technology [15-17] andmatrix-based secretion assays [18, 19].

In addition, automated systems for cell selection also exist and includeLaser enabled analysis and processing (LEAP) which destroys undesiredcells [20, 21] and automated colony pickers such as ClonePix fromGenetix and CellCelector™ from Aviso which select directly the highproducers according to their secretion levels.

Besides the various pros and cons of each method, their capacity for thenumber of cells that can be screened is limited, and significantly lowerthan that obtained by the e.g. Fluorescence-activated cell sorting(FACS).

SUMMARY OF INVENTION

The present invention comprises coupling protein of interest (POI)expression to that of a cell surface membrane protein/peptide markerlabeled with biotin mimetic or biotin acceptor peptides. Followingstaining with a fluorescently labeled biotin-binding protein, the cellsare sorted by an appropriate technique as discussed below.

The present invention relates, in one aspect, to a nucleic acid moleculecomprising:

-   -   (a) a first nucleic acid sequence encoding a signal peptide        linked at its C-terminal to    -   (b) a second nucleic acid sequence encoding a biotin mimetic        peptide (BMP) or a biotin acceptor peptide (BAP), linked at its        C-terminal to    -   (c) a third nucleic acid sequence encoding a polypeptide stretch        that allows the anchorage of the BMP or BAP to a cell membrane.

In a related aspect, the present invention provides a vector comprisingthe nucleic acid molecule as defined above.

The invention also relates to a protein encoded by the above nucleicacid molecules.

In another aspect, the present invention relates to a method for theselection of eukaryotic cells secreting a protein of interest (POI),comprising identifying cells presenting BMP on their cell surface,wherein the level of BMP presentation on the cell surface is correlatedwith the amount of POI secreted, the method comprising the steps of:

-   -   (a) transfecting cells with a vector comprising either        -   i. the nucleic acid molecule comprising the first, second            and third nucleic acid sequences above and a second nucleic            acid molecule comprising a POI-encoding nucleic acid            sequence; or        -   ii. a nucleic acid molecule comprising both nucleic acid            molecules on the same vector,    -   thereby establishing a stable pool of BMP transfected cells;    -   (b) labeling the BMP transfected cells with a detectable        biotin-binding moiety; and    -   (c) identifying and isolating transfected cells labeled with the        detectable biotin-binding moiety.

In another aspect, BAP is employed instead of BMP. In this case BirA andbiotin need to be added prior to labeling of the cells.

Improvement of the above system is obtained by fusing a polypeptideconferring selection resistance to the polypeptide stretch that allowsanchoring the BMP reporter. Applying selection pressure posttransfection selects transfected cells expressing the resistance gene.Since the selection resistance gene is fused to the BMP reporter gene,such application of selection is also efficient for high BMP expressingcells at early stages before sorting is done and may be efficient forselection of the highest POI producer cells in the population as well.

Thus, in certain embodiments, the polypeptide stretch that allows theanchorage of the BMP or BAP to a cell membrane is linked at its carboxylterminus to a polypeptide conferring selection resistance.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows vectors containing novel reporter genes for FACS clonedevelopment. The vectors were constructed for evaluation of novelreporter genes by FACS technology. BMP—Biotin mimetic peptide; SP—signalpeptide; TM—trans-membrane domain; PAC—puromycin resistance gene;C—peptide carrier motif; CD48-glycosyl phosphatidylinositol—GPI sequencetaken from CD48. hGH—human growth hormone; mCD59a —GPI sequence takenfrom mouse CD59a; hCMV—Human Cytomegalovirus immediate early promoter;SV 40—Simian vacuolating virus 40 promoter; IGF-1R—insulin like growthfactor-1 receptor; EMCV—Encephalomyocarditis virus; IRES—InternalRibosome Entry Site; CD164-Fc—an Fc fusion of sCD 164, GCSF—Granulocytecolony-stimulating factor; IL6—interleukin 6.

FIGS. 2A-C show the structures of reporter molecules on cell surface.Illustration of biotin mimetic peptide (BMP, panel A) and biotinacceptor peptide (BAP, panel B) fused to their membrane anchored carrieron cell surface. Anchorage to the membrane is obtained via glycosylphosphatidylinositol (GPI) or by a trans-membrane peptide (TM). BMP isstained directly with fluorescent streptavidin (F-SA). BAP is firstbiotinylated by biotin protein ligase (BirA), followed by staining withF-SA. FIG. 2C shows a biotin mimetic peptide (BMP) and puromycinN-acetyl transferase (PAC) fusion protein (BMP-PAC). Anchorage to themembrane obtained via a trans-membrane peptide (TM). PAC is used forpuromycin selection and BMP is stained directly with fluorescentstreptavidin (F-SA) for FACS selection and analysis.

FIGS. 3A-B is an illustration of a BMP-PAC expression vector (A)comprising the BMP-PAC reporter gene (B). The vector was constructed forevaluation of the BMP-PAC reporter gene by FACS technology.mCD59aSP—signal peptide from murine CD59a; BMP—Biotin mimetic peptide;Synthetic carrier—a carrier peptide of 60 amino acids; IGF1-R-TMdomain—trans-membrane domain from IGF 1 receptor; PAC-Puromycin N-acetyltransferase; mAb—monoclonal antibody; LC—light chain; HC—heavy chain,mCMVpr—murine immediate CMV-early-gene promoters.

FIG. 4 shows detection of BAP on cell surface with fluorescentstreptavidin (F-SA) by FACS. CHO-S cells, non-transfected and notstained (______, MFI-66). CHO-S cells, non-transfected, incubated withBirA and stained with F-SA (• • •, MFI-69). CHO-S transfected withplasmids expressing BAP-CD59a reporter gene, not incubated with BirA andstained with F-SA (- - -, MFI-139). CHO-S transfected with plasmidsexpressing BAP-CD59a reporter gene, incubated with BirA and stained withF-SA (- • -, MFI-24364).

FIGS. 5A-B show detection of BMP on cell surface with F-SA by FACS.CHO-S cells, non-transfected (• • •, MFI-27) or transfected (- • -,MFI-10516) with plasmid containing BMP-CD59a directly downstream to hCMVpromoter and stained with F-SA (A). CHO-S cells, non-transfected and notstained (______, MFI-66), non-transfected and stained with F-SA (• • •,MFI-36), transfected with plasmid containing BMP-CD59a downstream toEMCV IRES and either not stained (- - -, MFI-63) or stained with F-SA (-• -, MFI-1583) (B).

FIGS. 6A-C show CHO-S producer cells sorting with the BMP-CD59/F-SAsystem. Stable cells transfected with a vector containing sCD164-Fc (thePOI) and BMP-CD59a (the reporter gene for FACS sorting) (A), weresuccessively sorted three times according to their BMP levels indicatedby the F-SA (B), not labeled and not sorted (______, MFI-102), labeledbefore sortings (• • •, MFI-1734, PCD -1.59), after the first sort (- --, MFI-8804, PCD-4.60) after the second sort (- • -, MFI-16691,PCD-5.79), and after the third sort (- •• -, MFI-18995, PCD-7.34). Thecorrelation between fluorescence and productivity is shown (C). MFI,mean fluorescent intensity; PCD (specific productivity), Picogram perCell per Day; EMCV, Encephalomyocarditis virus; IRES, Internal ribosomeentry site; CD 164-Fc, CD 164 fused to Fc region.

FIGS. 7A-C show CHO-S producer cells sorting with the BMP-CD59/F-SAsystem. Stable cells transfected with a vector containing GCSF (the POI)and BMP-CD59a (the reporter gene for FACS sorting) (A), weresuccessively sorted three times according to their BMP levels indicatedby the F-SA (B), not labeled and not sorted (______, MFI-104), labeledbefore sortings (• • •, MFI-10406, PCD-3.83), after the first sort (- --, MFI-16661, PCD-6.03) after the second sort (- • -, MFI-16983,PCD-6.67), and after the third sort (- • • -, MFI-17676, PCD-6.63). Thecorrelation between fluorescence and productivity is shown (C).GCSF—Granulocyte colony-stimulating factor (Gene of interest).

FIGS. 8A-C show CHO-S producer cells sorting with the BMP-CD59/F-SAsystem. Stable cells transfected with a vector containing the gene forIL-6 (the POI) and BMP-CD59a (the reporter gene for FACS sorting) (A),were successively sorted three times according to their BMP levelsindicated by the F-SA (B), not labeled and not sorted (______, MFI-33),labeled before sortings (• • •, MFI-246, PCD- 0.4), after the first sort(- - -, MFI-1473, PCD-1.2) after the second sort (- • -, MFI-2812,PCD-1.7), and after the third sort (- •• -, MFI-3642, PCD-1.8). Thecorrelation between fluorescence and productivity is shown (C). IL-6-interleukin-6 (Gene of interest).

FIGS. 9A-C show CHO-S producer cells sorting with the BMP-P-GPI/F-SASystem. Stable cells transfected with a vector containing CD164-Fc (thePOI) and BMP-Peptide carrier (C)-GPI (the reporter gene for FACSsorting) (A), were successively sorted three times according to theirBMP levels indicated by the F-SA (B), not labeled and not sorted(______, MFI-33), labeled before sortings (• • •, MFI-773, PCD-0.8),after the first sort (- - -, MFI-9748, PCD-5.7) after the second sort (-• -, MFI-15492, PCD-9.5), and after the third sort (- • -, MFI-20908,PCD-10.8). The correlation between fluorescence and productivity isshown (C).

FIGS. 10A-C show CHO-S producer cells sorting with the BMP-P-GPI/F-SASystem. Stable cells transfected with a vector containing GCSF (the POI)and BMP-Peptide carrier (C)-GPI (the reporter gene for FACS sorting)(A), were successively sorted three times according to their BMP levelsindicated by the F-SA (B), not labeled and not sorted (______, MFI-113),labeled before sortings (• • •, MFI-2326, PCD-3.0), after the first sort(- - -, MFI-7866, PCD-7.1) after the second sort (- • -, MFI-17497,PCD-9.2), and after the third sort (- •• -, MFI-28679, PCD-13.0). Thecorrelation between fluorescence and productivity is shown (C).

FIGS. 11A-C show CHO-S producer cells sorting with theBMP-P-IGF-IR-TM/F-SA System. Stable cells transfected with a vectorcontaining CD 164-Fc (the POI) and BMP-Peptide carrier (C)-TM (thereporter gene for FACS sorting) (A), were successively sorted threetimes according to their BMP levels indicated by the F-SA (B), notlabeled and not sorted (______, MFI-33), labeled before sortings (• • •,MFI-680, PCD-1.1), after the first sort (- - -, MFI-4719, PCD-7.0) afterthe second sort (- • -, MFI-7522, PCD-9.9), and after the third sort (-•• -, MFI-9113, PCD-12.0). The correlation between fluorescence andproductivity is shown (C). TM-Trans-membrane peptide.

FIGS. 12A-C show CHO-S producer cells sorting with the BMP-P-IGF-IR-TM/F-SA System. Stable cells transfected with a vector containingGCSF (the POI) and BMP-Peptide carrier (C)-TM (the reporter gene forFACS sorting) (A), were successively sorted three times according totheir BMP levels indicated by the F-SA (B), not labeled and not sorted(______, MFI-113), labeled before sortings (• • •, MFI-4449, PCD-3.1),after the first sort (- - -, MFI-15764, PCD-7.1) after the second sort(- • -, MFI-26289, PCD-8.5), and after the third sort (- •• -,MFI-17367, PCD-12.0). The correlation between fluorescence andproductivity is shown (C).TM-Trans-membrane peptide.

FIG. 13 shows productivity of pools transfected with a plasmidcontaining BMP-PAC (the reporter gene for FACS sorting); and anti-IL22RAmAb (the POI). CHO-S cells, transfected with a vector containing BMP-PACand anti-IL22RA mAb in ProCHO5 were selected in 20 μg/ml puromycin and25 μM MSX. Samples were taken for productivity determination.

FIG. 14 shows CHO-S producer cells sorting with the BMP-PAC. Stablecells (3 replicates) transfected with a vector containing anti-IL22RA(the POI) mAb and BMP-PAC in ProCHO5 (the reporter gene for FACSsorting); were selected in 20 μg/ml Puromycin and 25 mM MSX and combinedto united pools. United pools were successively sorted three timesaccording to their BMP levels indicated by the F-SA. Pool not labeledand not sorted (______, MFI-67), labeled before sortings (• • •,MFI-4449, PCD-3.1), after the first sort (- - -, MFI-15764, PCD-7.1)after the second sort (- • -, MFI-26289, PCD-8.5), and after the thirdsort (- •• -MFI-17367, PCD-12.0).

FIG. 15 shows titers obtained in clones transfected with BMP-PACcontaining vector in medium containing ProCHO5. Accumulated titers ofanti-IL22RA MAb (the POI) following cloning in 96 well plates. Cloningwas done in 80% C6366+20% ProCHO5 and samples were taken for titerdetermination 13-15 days post cloning.

FIG. 16 shows specific productivity of clones transfected with BMP-PACcontaining vector in ProCHO5 medium. Cells were seeded at 0.5×10⁶cells/ml (10×10⁶ cells) in ProCHO5 medium in 50 ml tubes forproductivity measurements by ELISA. PCD-pg/cell/day.

FIG. 17 shows specific productivity of clones transfected with BMP-PACcontaining vector in ProCHO5 medium in the presence of 25 μM MSX. Cellswere seeded at 0.5×10⁶ cells/ml (10×10⁶ cells) in ProCHO5 mediumcontaining 25 μM MSX in 50 ml tubes for productivity measurements byELISA.

FIGS. 18A-B depict SDS PAGE /Western blot of mAb secreted by candidateclones in crude cell culture ProCHO-5 medium. Crude samples of candidateclones (100 ng of mAb per lane by ELISA) were separated by SDS-PAGE (10%BisTris) and transferred to nitrocellulose membrane. Detection was donewith goat anti-human IgG Fc horse-radish-peroxidase (HRP) (A), and goatanti-human kappa light chain (B), ECL was the substrate for the HRP. Themolecular weights of the markers (kDa) are shown to the left. Theidentification of the samples on the gels is as follows 1, MW marker; 2,Anti-IL22RA reference sample MSB0010074/C12; 3, Clone 2418-11; 4, Clone2418-14; 5, Clone 2418-15; 6, Clone 2418-39; 7, Clone 2418-45.

DETAILED DESCRIPTION OF THE INVENTION

Cell sorting by flow cytometry, e.g. FACS, requires a fluorsescentsignal. Since a protein of interest (POI) is usually not fluorescent, afluorogenic reporter gene product, the expression of which is linked tothe expression of the gene of interest (GOI) is required.

The biotin-streptavidin system of labeling cell surface marker isadvantageous for several reasons: 1) the system is applicable to manycell types with low background; 2) the affinity and specificity ofbiotin binding to streptavidin is very high; and 3) the system conformswith “animal component free” conditions, and does not require the use ofantibodies which may require a separate file to the regulatoryauthorities

Since biotin is not a protein or a peptide and therefore cannot beencoded by a specific DNA sequence, two strategies were taken: 1)utilization of a peptide that mimics biotin in its ability to bindstreptavidin (BMP) [22, 23], and 2) employment of a synthetic biotinacceptor peptide (BAP) [24] that can be specifically biotinylated by abiotin protein ligase (BPL), such as BirA, derived from bacterial origin[25, 26] that is either transfected into the cells as an encoding DNA[27-29] or added as a protein to the enzymatic mixture [24, 30, 31]. Thesynthetic BAP sequence [24] is specifically biotinylated similarly tothe longer natural sequence [26] by the enzyme BPL [26].

For enabling detection of the reporter with the F-SA on a cell surface,after being processed through the same secretion machinery used by thecell for processing and secretion of the POI, the reporter was designedto be bound to a cell membrane protein, processed by the secretorypathway in the ER and Golgi system. Since BMP and BAP do not havemembrane attachment sequences, according to the present invention thereare employed membrane bound sequences that are relatively short, can beeasily expressed, are not normally expressed on CHO cells, are not knownto be cytotoxic and do not have known negative biological effects on thecell.

Prediction of transmembrane domain sequences is well known in the art.It can be carried out, for example by using the TMHMM server v.2.0 [32]or for GPI anchored sequence by using the GPI modification siteprediction [33-36].

For the purpose of example only, two membrane anchors were chosen. Onewas the murine membrane protein CD59a which includes glycosylphosphatidylinositol (GPI), a small protein of 124 amino acids [37] thatwas reported to be expressed well in CHO cells and detected by FACS[38]. Additional reports in the literature on its bioactivity inprotecting cells from the complement lytic effect [39] does not indicatecytotoxicity risk. It is anchored to the external side of the cellmembrane by GPI [40] and it does not have an internal cytoplasmic domainthat could trigger signal transduction processes.

An additional membrane anchored protein is based on a synthetic peptidesequence of 60 amino acids and two potential N-glycosylation sites closeto its C-terminus, and either a GPI anchorage sequence, or atransmembrane (TM) sequence.

As mentioned above, in one aspect, the present invention provides anucleic acid molecule comprising: (a) a first nucleic acid sequenceencoding a signal peptide linked at its C-terminal to (b) a secondnucleic acid sequence encoding a biotin mimetic peptide (BMP) or abiotin acceptor peptide (BAP) linked at its C-terminal to (c) a thirdnucleic acid sequence encoding a polypeptide stretch that allows theanchorage of the BMP or BAP to a cell membrane.

In one embodiment, the nucleic acid sequences of (a) and (b) and/or (b)and (c), respectively, are linked by a nucleic acid sequence encoding apeptide linker.

In a further embodiment, the polypeptide stretch that allows theanchorage of the BMP or BAP to a cell membrane is CD59a which includesits GPI.

In another embodiment the polypeptide stretch which allows the anchorageof the BMP or the BAP to a cell membrane is the synthetic carrierpeptide of amino acid SEQ ID NO: 1 directly linked to CD48-GPI.

In yet another embodiment the polypeptide stretch which allows theanchorage of the BMP or the BAP to a cell membrane is the syntheticcarrier peptide of amino acid SEQ ID NO: 1 directly linked to atransmembrane peptide.

In a preferred embodiment, the transmembrane peptide is thetransmembrane peptide of IGF-1R of the amino acid sequence of SEQ ID NO:2.

As mentioned above, the BMP or BAP is linked at its N-terminus,optionally via a peptide linker, to a signal peptide. The signal peptidemay have any amino acid sequence that would direct the reporter to thecell membrane, an organelle membrane or outside of the cell. Thesequence may be easily predicted, for example by using algorithms astaught in the literature [41, 42].

Thus, in one embodiment, the BMP or BAP is linked at its N-terminus tothe signal peptide via a linker peptide, and the signal peptide is asignal peptide of a protein selected from the group consisting of anantibody, a cytokine, a hormone, a growth factor, a neurotransmitter, anenzyme, a receptor ligand, a toxin, or a functional fraction thereof.The signal peptide may also be the native or endogenous signal peptideof the protein of interest or the one of human growth hormone or ofCD59.

The ability to bio-select for transfected cells, to sort cellsexpressing the highest levels of the reporter gene and by that to selectthe cells producing the highest level of protein of interest may beimproved by fusing a polypeptide conferring selection resistance to thereporter in this way, the correlation between the resistance of thecells to the selection agent of choice, the expression of the reporterand the expression of the protein of interest is increased. Moreover,bio-selection is applied a short time post transfection and beforesorting, and this insures that most of the resistant population wouldexpress the selection resistance gene, the reporter gene and the GOI.

Thus, in a further embodiment, the polypeptide stretch that allows theanchorage of the BMP to a cell membrane is linked at its carboxylterminus to a polypeptide conferring selection resistance. Thispolypeptide is selected from the group consisting of anantibiotic-hydrolyzing enzyme, an antibiotic-scavenging protein, e.g.the Sh ble gene product that binds to and inhibits activity of Zeocin,and an antibiotic modifying enzyme such as a kinase. Alternatively,selection can be done by introduction of metabolic enzymes that areabsent in the cells or exist at very low levels and removal of thesemetabolites or the sources of the metabolites from the growth medium.Two known examples are the dihydrofolate reductase (DHFR) [43] and theglutamine synthetase (GS) [44] systems. Increased selection pressure andamplification that results in overproduction is obtained in the aboveexamples by addition to the growth medium of methotrexate for the DHFRsystem [45] or methionine sulphoximine for the GS system [46].

In one embodiment, the polypeptide stretch that allows the anchorage ofthe BMP or BAP to a cell membrane is linked to theantibiotic-hydrolyzing enzyme via a linker peptide.

In another embodiment, the antibiotic-inactivating enzyme is selectedfrom the group consisting of a puromycin-inactivating enzyme, such aspuromycin N-acetyl transferase (PAC) and an aminoglycoside (e.g.Geneticin) hydrolyzing enzyme, such as aminoglycoside3′-phosphotransferase; the antibiotic-scavenging protein is an Sh blegene product; and the antibiotics modifying enzyme is a hygromycinkinase.

The linkage between the BMP or BAP to the polypeptide stretch thatallows their anchorage to a cell membrane, the linkage between the BMPor BAP and their signal sequences and the linkage between thepolypeptide stretch and the polypeptide conferring selection resistancemay be direct or via a linker peptide.

In a preferred embodiment, the linker peptide consists of the amino acidsequence of isoleucine and proline or it consists of an amino acidsequence of SEQ ID NO: 4 ((G₄S)n(G4)_(m), wherein n is an integerselected from 1, 2, 3, 4, or 5, in particular it is 2 or 4; and m is aninteger selected from zero or 1.

In another embodiment, the nucleic acid molecule of the presentinvention is operably linked to a promoter, such as hCMV, capable ofdriving the transcription of the nucleic acid molecule, and isoptionally operably linked downstream to an EMCV IRES sequence.

As shown hereinafter in the examples, several plasmids were constructedwith different genes of interest (GOIs), and either the genes for BMP orBAP reporters fused to the membrane anchored carriers. The GOIs and thereporter genes were linked by an internal ribosome entry site (IRES). Inthose plasmids the GOI expression is driven by the powerful human CMV(hCMV) promoter and the reporter gene is located downstream to the IRES.The GOI expression may also be driven by any strong promoter, apart fromhCMV, such as, but not limited to murine CMV IE1 or murine CMV IE2,which were used herein below to drive the expression of the light andheavy chains of an IgG antibody. This bicistronic architecture dictatestranscription of both genes on the same mRNA [47].

In order to biotinylate the BAP sequence the cells were eitherco-transfected with the bacterial BirA expression vector [27] to obtainbiotinylation by the transfected cell itself [27-29, 31, 48], or the BAPwas biotinylated externally by addition of biotin and BirA in a specificreaction buffer [30, 49].

Eukaryotic cells, such as CHO-S cells were transfected with the plasmidsand stable pools were selected. Specific expression of BAP and BMP intransfected cells detected with fluorescent streptavidin (F-SA) wasdemonstrated without significant background. Furthermore, with the BMPas a reporter three consecutive sorts by FACS for high expressers weredone and significant improvement in the expression level of the GOIs wasobtained.

In a preferred embodiment, the plasmid, i.e. the nucleic acid moleculeof the invention, comprises a nucleic acid sequence that encodes for aBMP, in particular to BMP comprising the amino acid sequence of SEQ IDNO: 5, and specifically to BMP of SEQ ID NO: 5.

In one embodiment, the nucleic acid molecule comprises further nucleicacid sequences (plasmid MB-098) encoding the synthetic carrier peptideof the amino acid sequence of SEQ ID NO: 1 directly linked to thetransmembrane peptide of the amino acid sequence of SEQ ID NO: 2, thesignal peptide of CD59a of the amino acid sequence of SEQ ID NO: 6, andPAC of the amino acid sequence of SEQ ID NO: 7. In particular, thisnucleic acid molecule comprises the nucleic acid sequence of SEQ ID NO:8, which encodes for the BMP linked at its carboxyl terminus directly tothe synthetic carrier peptide that is directly linked at its carboxylterminus to the transmembrane peptide that is linked at its carboxylterminus via a (G₄S)₄ sequence to PAC, and said BMP is linked at itsamino terminus via an isoleucine-proline linker peptide to mouse CD59asignal peptide.

In another embodiment, the nucleic acid molecule comprises furthernucleic acid sequences (plasmids MB-070-MB-072) encoding (in addition toBMP of the amino acid sequence of SEQ ID NO: 5) for mouse CD59a of theamino acid sequence of SEQ ID NO: 9, and the signal peptide of CD59a ofthe amino acid sequence of SEQ ID NO: 6. In particular, this nucleicacid molecule comprises the nucleic acid sequence of SEQ ID NO: 10,which encodes for the BMP linked at its carboxyl terminus via a (G₄S)₂G₄sequence (SEQ ID NO: 4) to mouse CD59a and at its amino terminus via anisoleucine-proline linker peptide to the cognate CD59a signal peptide.

In still another embodiment, the nucleic acid molecule comprises furthernucleic acid sequences (plasmids MB-073 and MB-075) encoding (inaddition to BMP of the amino acid sequence of SEQ ID NO: 5) for thesynthetic carrier peptide of the amino acid sequence of SEQ ID NO: 1directly linked to the transmembrane peptide of the amino acid sequenceof SEQ ID NO: 2, and the signal peptide of CD59a of the amino acidsequence of SEQ ID NO: 6. In particular, this nucleic acid moleculecomprises the nucleic acid sequence of SEQ ID NO: 11, which encodes forthe BMP linked at its carboxyl terminus directly to the syntheticcarrier peptide that is directly linked at its carboxyl terminus to thetransmembrane peptide and said BMP is linked at its amino terminus viaan isoleucine-proline linker peptide to the mouse CD59a signal peptide.

In yet another embodiment, the nucleic acid molecule comprises furthernucleic acid sequences (plasmids MB-074 and 076) encoding (in additionto BMP of the amino acid sequence of SEQ ID NO: 5) for the syntheticcarrier peptide of the amino acid sequence of SEQ ID NO: 1 directlylinked to the CD48-GPI anchor peptide of the amino acid sequence of SEQID NO: 3, and the signal peptide of CD59a of the amino acid sequence ofSEQ ID NO: 6. In particular, this nucleic acid molecule comprises thenucleic acid sequence is of SEQ ID NO: 12, which encodes for a BMPdirectly linked at its carboxyl terminus to a synthetic carrier peptidethat is directly linked at its carboxyl terminus to CD48-GPI anchorpeptide and said BMP is linked at its amino terminus via anisoleucine-proline linker peptide to mouse CD59a signal peptide.

According to the present invention the reporter gene product and theprotein of interest may be encoded by nucleic acids on separateplasmids, or they may be encoded by nucleic acids on the same plasmid.

When the reporter gene product and the POI are encoded by nucleic acidson separate plasmids, the cells are cotransformed with both, the plasmidcontaining the reporter gene and the a second plasmid including the genecoding for the POI.

When one plasmid is employed, the present invention provides a nucleicacid molecule comprising (a) a first nucleic acid sequence encoding asignal peptide; (b) a second nucleic acid encoding a biotin mimeticpeptide (BMP) or a biotin acceptor peptide (BAP); (c) a third nucleicacid encoding a polypeptide stretch that allows the anchorage of the BMPor BAP to a cell membrane; and (d) a POI-encoding nucleic acid sequence.

In one embodiment, the POI-encoding nucleic acid sequence is operablylinked upstream to an EMCV IRES sequence or downstream to a promotercapable of driving the transcription of the nucleic acid molecule, orboth.

According to another embodiment several POI-encoding nucleic acidsequences may be present coding for several different subunits of amulti-subunit protein. Thus, for example, a cytokine of interest wouldbe encoded by a single POI-encoding nucleic acid sequence while an IgGor IgM would be encoded by two or three POI-encoding nucleic acidsequences, respectively.

In one embodiment, the POI-encoding nucleic acid sequence is onePOI-encoding nucleic acid sequence coding for a single polypeptide,while in another embodiment the at POI-encoding nucleic acid sequencecomprises two nucleic acid sequences coding for different subunits of anantibody. In many cases the protein of interest should be secreted tothe medium, and therefore in those cases it may be linked at its aminoterminus, optionally via a linker peptide, to a signal peptide.

The protein of interest may be any protein that can be expressed in aeukaryotic cell. As non-limiting examples, and just to illuminate whichproteins may be considered as protein of interest, the protein ofinterest is selected from the group consisting of an Fc-fusion product,an antibody, a cytokine, a hormone, a growth factor, a neurotransmitter,an enzyme, a receptor ligand, a sialomucin, a nuclear protein, aregulatory protein and a toxin, or a functional fraction thereof, i.e. afraction of the protein which itself is an antibody (for example anFab), a cytokine, a hormone, a neurotransmitter, an enzyme, a receptorligand, a sialomucin or a toxin. An Fc-fusion product is a proteinexpressed as a fusion to a signal peptide and the Fc fragment ofimmunoglobulin as the N-terminal or C terminal fusion partner, whichfacilitates expressing and secreting high levels of many different typesof POIs, as described above. The Fc domain helps to improve solubilityof hydrophobic proteins and provides a handle for easy detection andpurification of the fusion proteins; and it can be cleaved off bytreatment with protease, if desired.

It has been found in accordance with the present invention that highproducer cells that express various proteins of interest, such as aFc-fusion product (sCD164-Fc), an antibody (anti-IL22RA mAb), a growthhormone (granulocyte colony-stimulating factor (GCSF)) or a cytokine(IL6) and express high levels of these proteins can be efficientlyisolated by selecting for cells that express high levels of BMP on theirsurface.

In still another aspect, the present invention provides a method for theselection of eukaryotic cells secreting a protein of interest (POI),comprising identifying cells presenting BMP on their cell surface,wherein the level of BMP presentation on the cell surface is correlatedwith the amount of POI secreted, the method comprising the steps of:

(a) transfecting cells either with a vector comprising

-   -   (i) a nucleic acid molecule comprising the first, second and        third nucleic acid sequences above and a second nucleic acid        molecule comprising a POI-encoding nucleic acid sequence; or    -   (ii) a nucleic acid molecule comprising both nucleic acid        molecules under (i) above,        thereby establishing a stable pool of BMP transfected cells;

(b) labeling the BMP transfected cells with a detectable biotin-bindingmoiety; and

(c) identifying and isolating transfected cells labeled with thedetectable biotin-binding moiety.

In certain embodiments, each one of said at least one POI-encodingnucleic acid sequence is operably linked upstream to an EMCV IRESsequence and downstream to a promoter capable of driving thetranscription of the nucleic acid molecule, or both.

The cells used for the production of the protein of interest may be anyeukaryotic cell amenable to genetic manipulations, such as mammalian,plant, insect or yeast cells. Mammalian cells may be Chinese HamsterOvary (CHO) cells, baby mouse myeloma NS0 cells, hamster kidney (BHK)cells, human embryo kidney (HEK) cells, human retinal cells, COS cells,SP2/0 cells, WI38 cells, MRCS cells, Per.C6 cells. Plant cells can betobacco, carrot and rice cells. In one embodiment, the cells are CHOcells.

In certain embodiments, the cells are labeled by contacting them with adetectable biotin-binding protein or moiety selected from the groupconsisting of fluorescent avidin and fluorescent streptavidin and thelabeled cells are then identified and isolated by the means of a FACS.However, any method could be used to identify and isolate the labeledcells, for example, but not limited to, a method using beads coated witha biotin-binding moiety.

The method of the present invention provides for a high correlationbetween the amount of reporter gene product expressed on the surface ofthe cells and the amount of protein of interest expressed and secreted,and thus enables the selection of high producer cells by isolating cellsthat display high levels of reporter gene product. In particular, theselected eukaryotic cells are presenting higher amounts of BMP on theirsurface and are secreting larger amounts of protein of interest than thetransfected cells of the stable pool. In certain embodiments, theamounts of BMP on the surface of the selected eukaryotic cells and theamounts of protein of interest secreted by the selected eukaryotic cellsare larger by a factor ranging between 2 and 30-fold, i.e. at least by afactor selected from the group consisting of 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, and up to 30-fold-fold higher than the transfectedcells of the stable pool.

The membrane-bound reporter molecules of the present invention have beenfound, as shown hereinafter, to be efficient for sorting high producercells by FACS. Several important conclusions are derived from theresults: the BMP and BAP reporter proteins could be specificallydetected in the transfected cells; the background staining of labelednon-transfected cells was found to be negligible and therefore thesereporters are suitable for selection of the required cells (Example 3);The invention provides the ability to sort cells expressing the highestlevels of the reporter gene and by that to select the cells producingthe highest level of POI. The method was demonstrated with severalmembrane anchored scaffolds and several GOIs. Productivity was increasedfrom the pool stage to pool after three rounds of sorting cycles1.7-13.5 fold (see table 1) with different proteins and differentreporter structures. Additional sorting cycle along with cloning stepdone by the FACS according the same reporters expression levels may evenfurther increase the POI productivity levels in selected clones. Theseresults strengthen the validity of the system. The reporter structurefacilitates convenient staining with fluorescent-streptavidin. Thefluorescent-streptavidin used in these experiments was confirmed to beof non-animal derived origin which facilitates to use this reagent in aclone development process. An important development of the basicplatform was the fusion of the membrane-bound reporter molecule to thePAC selection gene; this construct was found to be very efficient forbioselection and sorting of high producer cells by FACS. It is importantto point out the following: the PAC selection resistance gene was foundto be active as a membrane bound protein even though naturally theenzyme is located in the cytoplasm; productivity increased duringsorting cycles up to ˜3 fold in pools (˜11 PCD) as compared with at thestart of the sorting cycles. Further increase was obtained in cloneswith values up to 6 fold in ProCHO5 medium (26 PCD). These resultsindicate that the expression of the reporter is tightly linked to thatof the POI.

The invention will now be illustrated by the following non-limitingexamples:

EXAMPLES

Materials and Methods.

Cells. CHO-S (GibcoBRL, Cat. #11619) cells adapted to CHO DHFR⁻ Mediumpowder, SAFC (Biosciences Cat. #C6614).

Reagents

AccuPrime Pfx Invitrogen DNA Polymerase Cat No. 12344-024

Acetic Acid (glacial) Merck Cat. #K28351556 (Germany). Acrylamidegels-NuPAGE; 10% Tris gel, Cat. #NP0301BOX, Invitrogen, (USA).

Agarose IBI Cat. #IB70042

Ampicillin—Sigma Cat. #A9518

Antibodies for ELISA: Capture: Goat anti-human IgG (H+L), Cat.#109-005-088 Jackson Immuno Research (USA). Detection: Goat anti-humanIgG Fab HRP, Cat. #109-036-098 Jackson Immuno Research (USA).

Antibody for Western blot: Staining: A) Goat anti-Human IgG Fc HRP,Jackson Cat. #109-036-098; B) Goat anti-Human kappa light chain,SouthernBiotech Cat. #2060-01 followed by Donkey anti-goat HRP, JacksonCat. #705-015-147.

Blotting paper GB002 Cat. #426677 Schleicher and Schuell (USA).

Bovine serum albumin (BSA), Bovostar. Bovogen Cat. #BSAS.01

Bovine serum albumin (BSA), Sigma Cat. #A-4503

Bromophenol blue, Merck, Cat. #2126169.

Dextran sulfate (Sigma, Cat. #D4911),

DHS5α competent bacteria, Life-Technologies Cat. #18263-012

DMSO, Merck, Cat. #K31630931

DNA ladder: 1 kb ladder for DNA, Biolabs Cat No. #3232L

DNA ladder: 100 by ladder for DNA, Biolabs Cat No. #3231 L

Electrophoresis sample buffer LDS, Invitrogen, Cat. #NP0007, (USA).

Ethanol, Merck Cat. 00983.1000.

FACS Accudrop Beads, BD, Cat. #MAB345249

Glucose, Sigma, cat. #G7021

Glutamine, Sigma, cat. #G5972

Hispeed plasmid Maxi Kit, Qiagen GmbH, Germany, Cat. #12663

HT Biological Industries Cat. #03-085-1C

Hydrochloric acid 37%, Merck, Cat. #1.00314.

Hydrochloric acid, Merck, cat. #UN-1789 1.00314.2500

LB+Ampicillin plates−Hy-Labs Cat. #PD178

LB medium for bacterial growth−Hy-Labs Cat. #BP302/400S.

LipofectAmine reagent, Gibco BRL Cat. #18324-020.

L-Methionine sulfoximine (MSX), Sigma Cat. #M5379 Luminogenic substrate:ECL Amersham kit, Cat. #RPN2109.

Methanol Merck Cat. #1.06009.2500

Mgc12-1M, SIGMA, Cat #M1028

NuPAGE MOPS running buffer, X20, InvitrogenCat. #NP0001

NuPage transfer buffer, Invitrogen Cat. #NP0006-1

NuPAGE Tris Glycin running buffer, X10, InvitrogenCat. #LC2675

Phenol red, Sigma, Cat. #P0290

Pluronic F-68, Sigma Cat. #P5556

Polyoxyethylenesorbitan Monolaurate (Tween 20), Sigma Cat #P-1379

Pre-Stained marker protein standard Cat. #LC5925, Invitrogen.

Protease inhibitor cocktail, Sigma Cat. #P8340

Pure cellulose nitrate membrane BA-85, Schleicher & Schuell, 78×90 mm,Cat. #401184.

Puromycin, InvivoGen Cat. #ant-pr-1

Reference samples Anti-IL22RA (MSB0010074/C12) was obtained from EMD(GVA) 5.9 mg/ml in PBS, pH 6.0).

Restriction enzymes were purchased from New England Biolabs. _oR-Phycoerythrin-conjugated Streptavidin (SA-PE), 0.2 mg/ml, BioLegend,Cat. #405203 (see CoO in 9.3 and CoA in 9.4).

Skim Milk powder, Fluka Cat. #70166

Sodium bicarbonate, Merck Cat. #6329

Sodium carbonate, Merck Cat. #6392

Sodium phosphate, Sigma, Cat. #S-3264

SYBR Safe DNA gel stain Invitrogen, Cat #S33102, 3□/Gel

TMB Savion diagnostics, Cat. #1928

Tween20 (Polyoxyethylene-Sorbitan Monolaurate)-Sigma Cat. #P-1379

Water R.O. (ITL)

Whatman 3 mm, Whatman Cat. #3030917

Solutions

Bleach 1% -FACSClean, Becton Dickinson cat. #340345.

PBS (ITL preparation, BR R0450V01).

PBS with 0.1% Pluronic acid.

Culture Media

CHO DHFR-cloning Medium, SAFC Bioscinces Cat. #C6366, supplemented with4 mM L-Glutamine and 15 mg/L Phenol red, Sigma, Cat. #P0290.

ProCHO5 medium, Lonza Cat. #BE12-766Q, supplemented with 4 mML-Glutamine and 15 mg/L Phenol red, Sigma, Cat. #P0290.

Minimum Essential Medium Eagle, Sigma, cat. #M2279.

Methods

Construction of DNA expression vectors. All vectors were constructedutilizing standard molecular biology techniques as taught for example inF. M. Ausubel et al.[F. M. Ausbel, 2009 #132}.

Preparation of plasmid DNA. Plasmid DNA was isolated using QIAGENHispeed plasmid Maxi Kit according to the procedure described by themanufacturer.

DNA sequencing. The DNA fragments prepared by a contract firm (GeneArt,Germany) and cloned into the vector by standard procedures weresequenced at Hy-Labs, Israel. DNA sequencing was performed by the fullyautomated 16 Capillary ABI Prism 3100 Genetic Analyzer. The sequence wasanalyzed in-house utilizing the Sci-Ed General software (Clone managersoftware, version 7.01 and Align plus 5, version 5.01).

Transfection of CHO-S cells. CHO-S cells in were thawed and cultured inProCHO5 serum free medium (Lonza, Cat. #BE12-766Q) supplemented withHypoxanthine 13.61 mg/L and Thymidine 3.88 mg/L (HTx1, BiologicalIndustries Cat. #03-085-1B). Cells were grown in suspension in filtertubes 50 ml Bioreactor (TPP, Cat#87050), 37° C., humidified and shakenat 320 RPM. Two days prior transfection, the cells were seeded at aconcentration of 0.2×10⁶ cells/ml in Erlenmeyer, 500 ml, with cap(Corning, Cat. #431145). The cells were transfected by LipofectAmine(GibcoBRL Cat. #18324-020). On the day of transfection, the cells werewashed; resuspended and 10×10⁶ cells were seeded in 4 ml MEM (Sigma,Cat. #M2279) in Erlenmeyer 125 ml with filter cap (Corning, Cat.#431143). For each transfection with single plasmid, 20 μg linearizedvector (MB-098) were used. The final DNA volume was adjusted to 100 μlin MEM. Subsequently, 100 μl LipofectAmine were added and incubated for45 minutes at room temperature. The DNA-LipofectAmine mix was then addedto the cells and incubated for 4 hours at 37° C., 5% CO₂ in a shakingincubator at 45 RPM. At the end of this incubation period the cells werespun down and medium was replaced with 20 ml fresh ProCHO5 (Lonza, Cat#BE12-766Q) supplemented with Hypoxanthine 27.22 mg/L and Thymidine 7.76mg/L (HTx2, Biological Industries Cat. #03-085-1B) in Erlenmeyer 125 mlwith filter cap (Corning, Cat. #431143). The flask was incubated at 37°C. in a shaking incubator at 125 RPM for 72 hours.

Seventy two hours post transfection, the cells were collected,centrifuged and resuspended in 20 ml ProCHO5 medium supplemented with 20μg/ml Puromycin (Invivogen, Cat. #ant-pr-1), 25 μM MSX (Sigma, Cat.#M5379) and 100 μg/ml dextrane sulfate (Sigma, Cat. #D4911) Under theseselective conditions, only cells expressing the PAC gene could survive.

In vitro biotinylation. Cells cultured in animal component free medium(ACFM) were washed in PBS (phosphate buffer saline) with 0.1% Pluronicacid F-68 (Sigma Cat. #P5556) and 5 mM MgCl2 (Sigma, Cat. #M1028,) andthen 2×10⁶ cells were incubated in PBS with 0.1% Pluronic acid F-68(Sigma, Cat. #P5556,), 5 mM MgCl2 (Sigma, Cat. #M1028), 0.06 mM BirA(Avidity, Cat. #BirA500) 1 mM ATP (Sigma, Cat. #A6419,) and 10 mM biotin(Sigma, Cat. #B4639,) for one hour in room temperature. After incubationthe cells were washed with PBS with 0.1% Pluronic acid F-68 (Sigma, Cat.#P5556,) and 5 mM MgCl2 (Sigma, Cat. #M1028).

Labeling with fluorescent streptavidin (F-SA). Cells (2×10⁶) expressingBAP that were either biotinylated in vivo or in vitro or cellsexpressing BMP, were washed with PBS with 0.1% Pluronic acid F-68(Sigma, Cat. P5556) and then incubated in PBS+0.1% pluronic acid F-68+R-Phycoerythrin-conjugated Streptavidin (SA-PE) (Jackson, Cat.016-110-084) (F-SA) diluted 1:100 and incubated for 30 minutes at 37° C.with shaking at 80 RPM. Cells were then washed in PBS with 0.1% Pluronicacid F-68 and loaded on the FACSAria for analysis.

Analysis by FACS. Cells (2×10⁶) expressing BMP were washed with PBScontaining 0.1% Pluronic acid F-68 (Sigma, Cat. P5556) and thenincubated in PBS+0.1% pluronic acid F-68+R-Phycoerythrin-conjugatedStreptavidin (BioLegend, Cat. #405203) (F-SA) diluted 1:100 andincubated for 30 minutes at 37° C. with shaking at 80 RPM. Cells werethen washed in PBS with 0.1% Pluronic acid F-68 and loaded on theFACSAria for analysis.

Cell propagation. Cell cultures were maintained in ACFM as follows:Cells were seeded into 50 ml tubes at a concentration of 0.2×10⁶ cell/mlin 25 ml volume and incubated at 37° C. on an orbital shaker φ25 mm at320 rpm. Twice a week, cell number and viability were measured. Theculture was passaged by centrifugation at 100 g for 5 minutes at 4° C.and cell pellet was then re-suspended in fresh pre-warmed ACFM.

Cell Propagation and Productivity in ACFM

Cell culture maintenance. Cell cultures were maintained in ACFM asfollows: Cells were seeded into T-80 flasks at a concentration of0.2×10⁶ cell/ml and incubated at 37° C. on an orbital shaker at 45 rpm.Twice a week, cell number and viability were measured. The culture waspassaged by centrifugation at 100 g for 5 minutes at 4° C. and cellpellet was then re-suspended in fresh pre-warmed ACFM.

Tissue culture flasks 25 cm2 were seeded with 8-10 ml medium andincubated on an orbital shaker f25 mm at 55 rpm. Tissue culture flasks80 cm2 were seeded with 20-30 ml medium and incubated on an orbitalshaker f25mm at 45 rpm.

Cell productivity in ACFM. For specific productivity (PCD) in ACFM,cells were seeded in the specified ACFM at a concentration of 0.5×10⁶cells/ml, in a 50 ml tube and incubated at 37° C. on an orbital shaker(320 rpm) for 24 hours. Medium was then sampled and productconcentration was determined by ELISA. The calculation was done bydividing the 24 hours titters by the average concentration of cells atseeding and after 24 hours of the experiment.

${PCD} = \frac{T}{\frac{{Ci} + {Ce}}{2}}$ PCD-pg/cell/dayT-titer(pg/ml)

Ci—cell concentration at seeding (cells/ml)

Ce—cell concentration after 24 hrs (cells/ml)

Cell Sorting. For FACS sorting transfected cells from united pools inProCHO5 medium supplemented with 20 μg/ml puromycin and 25 μM MSX wereused. For each sort, approximately 60×10⁶ cells were cultured andlabeled with F-SA. Cells to be sorted were labeled in PBS containing0.1% pluronic acid F-68 and F-SA diluted 1:100 at a concentration of4×10⁶cells/ml in T80 flasks and incubated, 1 hour, at 37° C. in shakingincubator at 80 rpm. After labeling, cells were collected, washed twicein PBS+0.1% Pluronic acid F-68, and re-suspended at a finalconcentration of 10×10⁶ cells/nil in PBS+0.1% Pluronic acid F-68 forbulk sorting. The top 4% fluorescent cells were gated on an FSC/PE dotplot for sorting with the FACSAria flow cytometer in the ‘Single cell’precision mode. The sorted cells were seeded in ProCHO5 mediumsupplemented with 20 μg/ml puromycin and 25 μM MSX and allowed to growuntil cell viability was >=90% and viable cell number was ≧70×10⁶. Atthis stage analysis of fluorescence and the next sorting cycle weredone. Overall, three successive sorts were done.

Cloning by FACS ACDU. Cloning was done by the Automated Cell DepositionUnit (ACDU) device of the FACSAria cell sorter, of cells growing inProCHO5 containing 20 μg/ml puromycin and 25 μM MSX. 2×10⁶ cells werecollected, washed twice in PBS+0.1% pluronic acid and labeled in 0.5 mlof SA-PE (BioLegend) at a concentration of 2 μg/ml (dilution 1:100). Thecells were incubated in 24 wells plate for 30 minutes at 80 rpm in a 37°C. Following labeling, cells were collected, washed twice in PBS+0.1%Pluronic, and re-suspended in 4 ml of PBS+0.1% pluronic acid (cellconcentration of ˜0.1-0.2 cell/ml). The top 2.5% fluorescent cells werecloned by the ACDU, in the “Single Cell” precision mode, into 96 wellplates containing 180 μl/well of 80% Sigma C6366 and 20% ProCHO5 ACFMmixture for cells that were cultured in ProCHO5. The plates wereanalyzed by Cellavista at day 0 and then every 2-3 days for detection ofwells with single colony. Two weeks after cloning, supernatants fromwells in which colony growth was detected, were sampled and assayed byELISA. Wells containing >1 colony per well were omitted. Cells werepicked from the wells with the highest titers and transferred first toT25 flasks containing 4 ml of 50% Sigma C6366 and 50% ProCHo5 mediummixture without shaking. Three to five days later 2-4 ml of the 100%ProCHO5 medium was added to the T25 flasks and the cells were incubatedwith shaking. One to three days later 8 ml of ProCHO5 were added and thesuspension was transferred to T80 flasks (total of 87.5% ProCHO5 and12.5% C6366). One to two days later 10 ml of fresh medium were added tocells. Then cells were seeded in 25 ml fresh medium at 0.2×10⁶ cells/mlfor another growth cycle. For determining specific productivity thecells were seeded at concentration of 0.5→10⁶ cells/ml for 24 hours at37° C. in 50 filter tubes at 320 rpm.

ELISA for sCD164-Fc. The following procedure was employed:

1. Microtiter plates were coated with 0.5 mg/ml with monoclonal antibodyto CD164 in PBS and incubated overnight at ˜4° C.

2. The plates were washed three times with washing buffer (PBScontaining 0.05% of Tween 20).

3. The plates were blocked with blocking buffer (PBS containing 0.5%I-Block), 200 μl/well, for 1 hour at 37° C. with shaking.

4. After blocking, the plates were washed three times with washingbuffer and 100 ml aliquots of tested samples, standard curve (0.78-50ng/ml) and check samples in assay buffer (PBS containing 0.25% I-Blockand 0.05% of Tween 20), were added to the plates and incubated for 60min at 37° C. with shaking.

5. Plates were washed again three times with washing buffer, and 100 mlof the second antibody to the Fc portion (HRP cojugate F(ab′)2 fragmentgoat anti human diluted 1:40,000 in assay buffer) were added to eachwell.

6. The plates were incubated for 60 min at 37° C. with shaking.

7. Plates were washed three times with washing buffer and 100 ml ofsubstrate solution (TMB) were added to each well and the plates wereincubated 20 min at room temperature (without shaking).

8. The reaction was stopped by adding 50 μl/well of stop solution (4NHCl).

9. The absorbance was measured at A492 nm in an ELISA reader.

10. Standard solutions were prepared by serial dilutions of the Std.SCD164-Fc from 293 HEK cells to give a standard curve range from 0.78 to50 ng/ml (linear range was between 0.78-12.5 ng/ml) in assay buffer. Asample of crude harvest from the SCD164-Fc pool was diluted with assaybuffer to obtain ˜10 ng/ml and used as a check sample.

11. The optical density data results were processed and resultscalculated by the Magelan software.

12. The dilution of samples, preparation of standard curve dilutionseries, and distribution of samples on the plate was performed by arobotic sample processor.

ELISA for GCSF. The following procedure was employed:

1. Microtiter plates were coated with 1 μg/ml with monoclonal antibodyto GCSF in PBS and incubated overnight at ˜4° C.

2. The plates were washed three times with washing buffer (PBScontaining 0.05% of Tween 20).

3. The plates were blocked with blocking buffer (PBS containing 1% BSA),200 μl/well, for 1 hour at 37° C. with shaking.

4. After blocking, the plates were washed three times with washingbuffer and 100 ml aliquots of tested samples, standard curve samples andcheck samples were added to the plates and incubated for 60 min at 37°C. with shaking.

5. Plates were washed again three times with washing buffer, and 100 mlof the detection biontinylated antibody anti-human GCSF solution, wereadded to each well.

6. The plates were incubated for 60 min at 37° C. with shaking.

7. The plates were incubated with a second antibody, Streptavidin HRP,following the same procedure described in steps 5-6 above.

8. Plates were washed three times with washing buffer.

9. 100 ml of substrate solution (TMB) were added to each well and theplates were incubated 20 minutes at room temperature (without shaking).

10. The reaction was stopped by adding 50 μl/well of stop solution (3NHCl).

11. The absorbance was measured at 492 nm in an ELISA reader.

12. Standard solutions were prepared by serial dilutions of the Standardrecombinant human GCSF to give a standard curve of 1.56-100 ng/ml(linear range was between 3-50 ng/ml).

13. The optical density data results were processed and resultscalculated by the Magelan software.

14. A robotic sample processor performed the dilution of samples,preparation of standard curve dilution series, and distribution ofsamples on the plate.

ELISA for IL-6 The following procedure was employed:

1. Microtiter plates were coated with 1 mg/ml with monoclonal antibodyto IL6 in PBS and incubated overnight at 4° C.

2. The plates were washed three times with washing buffer (PBScontaining 0.05% of Tween 20).

3. The plates were blocked with blocking buffer (PBS containing 1% BSA),200 μl/well, for 1 hour at 37° C. with shaking.

4. After blocking, the plates were washed three times with washingbuffer and 100 ml aliquots of tested samples, standard curve samples andcheck samples were added to the plates and incubated for 60 min at 37°C. with shaking.

5. Plates were washed again three times with washing buffer, and 100 mlof the detection antibody: biotinylated anti-IL6, were added to eachwell.

6. The plates were incubated for 60 min at 37° C. with shaking.

7. The plates were incubated with a second antibody, Avidin-HRP,following the same procedure described in steps 5-6 above.

8. Plates were washed three times with washing buffer.

9. 100 ml of substrate solution (TMB) was added to each well and theplates were incubating 20 min at room temperature (without shaking).

10. The reaction was stopped by adding 50 μl/well of stop solution (3NHCl).

11. The absorbance was measured at 450 nm in an ELISA reader.

12. Standard solutions were prepared by serial dilutions of the IL6reference standard give a standard curve of 31.75-2000 pg/ml (linearrange was between 31.75-500 pg/ml).

13. The optical density data results were processed and resultscalculated by the Magelan software. A robotic sample processor performedthe dilution of samples, preparation of standard curve dilution series,and distribution of samples on the plate.

Collecting Anti-IL22RA cell culture harvest for analysis. Producer Cells(0.5×10⁶) were seeded in 20 ml ProCHO5 medium in filter tube 50 andcultured for 24 hours at 37° C. on a shaker at 320 rpm. The harvest wascentrifuged and filtrated throughout 0.22 μm filter. The clarifiedsupernatant was analyzed by ELISA assay followed by Western blot assay.

ELISA for Anti-IL22. The following procedure was employed:

1. Microtiter plates were coated with 100 μl per well of 2.0 μg/ml Goatanti-human IgG (H+L) in coating buffer and incubated overnight at ˜4° C.in humid box. The plates can be stored in −20° C. for 3 months after O/Nincubation.

2. The plates were washed four times with washing buffer (PBS containing0.05% of Tween 20).

3. The plates were blocked with blocking buffer (BSA 1% in PBS-T 0.05%),200 μl/well, for 1 hour at RT.

4. After blocking, the plates were washed four times with washing bufferand 100 μl aliquots of tested samples, standard curve (1.56-100 ng/ml)and check samples in assay buffer (Milk 1% in PBSx1). The plates werecovered with a plate sealer and incubated for 60 min at 37° C. noshaking.

5. Plates were washed again four times with washing buffer, and 100 μlof the second antibody goat anti human IgG Fab HRP diluted 1:100,000 inassay buffer) were added to each well.

6. The plates were incubated for 60 min at 37° C.

7. Plates were washed four times with washing buffer and 100 μl ofsubstrate solution (TMB) were added to each well and the plates wereincubated 15-20 min at room temperature (without shaking).

8. The reaction was stopped by adding 100 μl/well of stop solution (1NHCl).

9. The absorbance was measured at A450 nm in an ELISA reader.

10. Standard solutions were prepared by serial dilutions of the Std.Anti-IL22RA MSB0010074/C12 in PBS, pH 6.0 to give a standard curve rangefrom 1.56 to 100 ng/ml (linear range was between 1.56 to 50 ng/ml) inassay buffer. A sample of crude harvest from the relevant pool wasdiluted with assay buffer to obtain ˜25 ng /ml and used as a checksample.

11. The optical density data results were processed and resultscalculated by the Magelan software.

12. The dilution of samples, preparation of standard curve dilutionseries, and distribution of samples on the plate was performed by arobotic sample processor.

SDS-PAGE/Western blot analysis. Clarified supernatant samples ofanti-IL22RA clones in ProCHO5 medium was diluted in SDS PAGE samplebuffer. Samples of anti-I122RA clones (0.1 μg per lane by ELISA) wereseparated on SDS-PAGE 10% Bis Tris gels under non-reducing conditions.The pre-stained MW protein standard (15 mcl) was loaded on the gel aswell. Electrophoresis was performed at constant voltage (100 V) for ˜2hr with a Novex Xcell SureLock Mini-Cell electrophoresis system. At theend of the SDS PAGE run the proteins were transferred from the gel to anitrocellulose membrane in a blotting module with transfer buffer usingpower supply adjusted to 35 volt for 1 hour. The membrane was washedwith PBS-0.05% Tween 20 for 5 min, followed by incubation blockingbuffer over night at 4° C. The membrane was incubated with the followingantibodies diluted in working buffer, for 2 hours at room temperaturewith shaking: a) Goat anti-Human IgG Fc HRP b) Goat anti-Human kappalight chain followed by Donkey anti-goat HRP. The membrane was thenwashed in PBS-0.05% Tween 20 three times for 5 min each time. The bandswere visualized by incubation in ECL reagent for 1 min following byexposure of a film in an AFP X-Ray Film Processor “Mini-Medical”developer. After developing the film was scanned.

Example 1 Platform Design

The platform design for evaluation of BMP and BAP expression levels oncell surface was created first with the reporter protein located on anindividual expression cassette to insure sufficient expression andpermit expression evaluation (FIG. 1; vectors MB-065, MB-066 & MB-067).After verification of reporter protein expression and detection on cellsurface by FACS, new BMP containing vectors were constructed with atight linkage between the BMP based reporter genes and the GOIs in abicistronic mRNA. The plasmids were composed of the GOIs driven by thepowerful human CMV (hCMV) promoter and the reporter gene was locateddownstream to the EMCV IRES. This architecture dictates transcription ofboth genes on the same mRNA [47] (FIG. 1; vectors MB-070-MB-076).

The reporter molecule contained a membrane bound protein or peptide anda reporter moiety (BMP or BAP). The first membrane bound protein wascomposed of the CD59a from mouse, which is a small protein of 101 aminoacids [37] anchored via its C terminal end to the external side of thecell membrane by glycosyl phosphatidylinositol (GPI) [40] (vectorsMB065, MB066, MB-070-MB-072 in FIG. 1). The nucleic acid sequence of theBMP-CD59a construct is as set forth in SEQ ID NO: 10). Alternatively, asynthetic peptide of 60 amino acids was created (SEQ ID NO: 1), with twopotential N-glycosylation sites, that was anchored to the cell membranevia either a TM domain from mouse IGF-I receptor (SEQ ID NO: 2; vectorMB-073 & MB-075; FIG. 1) or a GPI anchorage domain from CD48 (Cysresidues were replaced by Ser; SEQ ID NO: 3; vectors MB-074 & MB-076;FIG. 1). Prediction of TM domain sequence was done by TMHMM serverv.2.0, [32] and GPI anchored sequence was done by GPI modification siteprediction.[33-36] The N terminal end of the membrane protein or peptideor the GPI anchored signal are bound via a linker to either the BMPcoding for amino acid sequence of CHPQGPPC [22, 23] (SEQ ID NO: 5) orthe BAP coding for amino acid sequence of GLNDIFEAQKIEWHE [24] (SEQ IDNO: 13).

Cells transfected with BMP containing vectors were directly stained withfluorescently labeled streptavidin (F-SA, FIG. 2A). Staining of the BAPexpressing cells was required prior to biotinylation of this reporterpeptide. This was achieved by either co-transfection of the cells withCHO optimized BirA sequence [27] to obtain in-vivo biotinylation by thecell itself (data not shown) [27-29, 31, 48] or by in-vitrobiotinylation of BAP on the cell surface by adding the bacterial enzymeBirA exogenously [30, 49] (FIG. 2B).

The platform was expanded in order to stringently select for cellsproducing high levels of the gene of interest by fusing BMP with the PACresistance gene. The BMP-PAC permits puromycin selection aftertransfection followed by FACS selection according to the BMP levels. Aplasmid was constructed for the expression of a model antibody, composedof two expression cassettes one for the heavy chain and one for thelight chain. The heavy chain expression is driven by the powerful murineCMV IE2 (mCMV IE2) promoter and the reporter gene is located downstreamto an EMCV IRES. The light chain expression is driven by the murine CMVIE1 (mCMV IE1) promoter and a QSy (glutamine synthetase) selectionmarker is located downstream to another EMCV IRES. This architecturedictates transcription of the heavy chain and BMP-PAC genes on a singlemRNA and the transcription of the light chain and the QSy on anothermRNA[47] (FIG. 3; vector MB98).

The reporter-selection molecule contains: the BMP preceeded by a signalpeptide derived from murine CD59a molecule. The BMP is fused on its Cterminus to a synthetic peptide of 60 amino acids, with two potentialN-glycosylation sites. This synthetic carrier is linked to a TM domainfrom mouse IGF-I receptor. The TM domain is linked to the PAC selectionresistance gene via a short linker (FIG. 2C, FIG. 3; vector MB-098).Prediction of TM domain sequence was done by TMHMM server v.2.0. The BMPis encoded by the amino acid sequence of SEQ ID NO: 5).

PAC in this specific construct is attached to the cell membrane.Therefore PAC has to contain efficient N acetylation activity as amembrane bound protein, which is different from its natural cytoplasmicstate.

Example 2 Construction of Expression Vectors

For this project three sets of vectors were constructed at differentstages of the project:

In the first set the biotin mimetic peptide (BMP), Biotin acceptorpeptide (BAP) and BirA genes were cloned into vectors directlydownstream to the hCMV promoter. These vectors were initially used totest the BMP and BAP expression level on cell surface and the ability todetect these markers with fluorescent streptavidin (F-SA) by FACS, andin later experiments as control vectors. The second set of vectorscontained the BMP-CD59-GPI cassette downstream of an EMCV IRES thuslinking the FACS selection marker to the GOI in a tighter and directmanner than in the vectors used before. The third set contained asynthetic peptide carrier instead of the mouse CD59a (which includesGPI), anchored to the cell membrane via GPI or TM domains (FIG. 1). As aselection marker the PAC gene was inserted in the vector downstream tothe SV40 promoter, resulting in cytosolic PAC in the transfected cells.

All vectors were constructed according to known procedures as taught forexample in F. M. Ausubel et al.[50].

Example 3 Evaluation of the Reporter Molecules for Sorting High POIProducer Cells

3.1 Expression and detection of the reporter molecules on cell surface.Generation of cells expressing GOI and BMP or GOI and BAP or GOI and BAPand BirA was done by LipofectAmine transfections of plasmids into CHO-Scells followed by selection with puromycin or puromycin and zeocin incells co-transfected with BirA containing plasmid.

The stable pools were analyzed for expression levels of the reportergenes. BAP level was analyzed following biotinylation by BirA addedexogenously and then labeled with F-SA (FIG. 4) or biotinylationintracellularly in those cells co-transfected with BirA, followed bylabeling with F-SA (data not shown).

BMP level was analyzed directly by binding of F-SA. The results showspecific fluorescence signal of the reporter molecules on thetransfected cells with no significant background on non-transfected andnon-labeled cells (FIG. 5A).

In FIG. 5, CHO-S cells, non-transfected and transfected with plasmidscontaining BMP-CD59a directly downstream to hCMV promoter (A) ordownstream to EMCV IRES (B) were either stained, or not stained withF-SA and analyzed for their fluorescent labeling levels by FACS. Thefluorescence level of the streptavidin-stained BMP (FIG. 5) is lowerthan that of the streptavidin-stained BAP (FIG. 4).

Possible reason for the difference in the fluorescent labeling intensityof cells stained with BMP and BAP could reside in the significantlylower affinity of the biotin mimetic peptide to streptavidin vs. naturalbiotin in the case of BAP (several order of magnitudes). In spite ofthis difference, high staining intensity of BMP expressing cells wasobtained, significantly above background to enable sorting of thelabeled cells by FACS.

Two peaks with low and high fluorescence are seen in the transfected andlabeled cells (FIGS. 5A-B; curves - •• -). Those two peaks probablyindicate low and high BMP expressers. It is speculated that the lowexpressers express also low levels of PAC even though the PAC is locatedon separated expression cassette. Nevertheless the PAC level issufficient for the cells to survive under the puromycin selectionpressure. 3.2 Sorting and generation of high producer cells. Cells weretransfected with vector containing three different GOIs (sCD164-Fc,GCSF, IL-6) and the chimera of BMP-CD59a (vectors MB-070, MB-071,MB-072), BMP-Peptide carrier-GPI (vectors MB-074 & MB-076) orBMP-Peptide carrier-TM (vectors MB-073 & MB-075) (FIGS. 6-12) where theGOI and BMP are located on the same bicistronic expression cassetteseparated by an EMCV IRES. The stable pools generated by bio-selectionwith puromycin (expressed on a different cassette) were analyzed for theexpression levels of the reporter molecules by FACS and the GOIs byspecific ELISA.

Two peaks with low and high fluorescence are seen in the transfected andlabeled cells (FIGS. 6-12; curves - • • -). Those two peaks indicate lowand high BMP expressers. It is speculated that the low expressersexpress also low levels of PAC. It is probable that the PAC level issufficient for the cells to survive under the puromycin selectionpressure but the BMP expression and possibly also the POI level of thosecells is low.

In all cases (FIGS. 6-12) significant levels of BMP as well as the GOIswere detected. The cells were labeled with F-SA and the 4% highestfluorescent cells were sorted. The sorted cells were then propagated andanalyzed for BMP and GOIs levels. The sorting procedure and analyseswere successively repeated three times. In every sort the highest BMPexpressed population was enriched, and as a consequence, theproductivity level of the GOI was elevated (A positive correlationbetween the GOI and BMP expression levels was observed (FIG. 6 to FIG.12).

Example 4 Construction of Expression Vectors Encoding for Reporter-PACFusion Product

4.1 Construction of vector MB-098 (FIG. 3)—Vector MB-098 was constructedaccording to known procedures as taught for example in F. M. Ausubel etal.[50].

4.2 Generation of stable pools containing BMP-PAC reporter, evaluationof PAC activity and productivity analysis. CHO-S cells transfected inProCHO5 with the BMP-PAC containing vector were subjected to selectionwith 20 μg/ml Puromycin (Invivogen, Cat. #ant-pr-1) and 25 μM MSX (SigmaCat. #M5379). Under these conditions stable cells emerged whichindicates that PAC is active in the membrane bound form. Stable poolsexpress the anti-IL22RA mAb with specific productivity of 8.5-9.4 PCD(FIG. 13).

4.3 Sorting and generation of high producer pools containing BMP-PACreporter. The stable cells were analyzed and sorted by FACS in ProCHO5medium (FIG. 14). Analyses show that BMP is detected in transfectedpools. Two subpopulations are seen before sorts, which express high andlow BMP levels. Consecutive sorts performed by selection of the 4%highest BMP expresser cells resulted in PCD elevation which wascorrelated with the BMP levels. Summary of results are seen in Table 1.

TABLE 1 Summary of sorting results using the BMP and BMP-PAC*. MFIbefore MFI after PCD before PCD after Reporter GOI sorts sorts sortssorts BMP-CD59** sCD164-Fc 1734 18995 1.6 7.3 BMP-CD59** GCSF 1040617676 3.8 6.6 BMP-CD59** IL-6 246 3642 0.3 1.8 BMP-IGF1-R- GCSF 444947367 3.1 12.0 TM** BMP-CD48-GPI** GCSF 2326 28679 3.0 13.0BMP-IGF1-R-TM** sCD164-Fc 680 9113 1.1 12.0 BMP-CD48-GPI** sCD164-Fc 77320908 0.8 10.8 BMP-IGF1-R-TM-PAC** Anti- 4449 17367 3.1 12.0 IL22RA mAb*Shown are the results of the mean fluorescence intensity (MFI) and POIlevels before and after three sorts. **Transfected cells were culturedin ProCHO5 medium

Stable cells (3 replicates) transfected with a vector containinganti-IL22RA mAb and BMP-PAC in ProCHO5 (FIG. 14) were selected in 20μg/ml Puromycin and 25 mM MSX and combined to united pools. The cellswere analyzed for BMP level (red line). Pools were successively sortedthree times according to their BMP levels labeled with F-SA. After eachsorting cycle the cells were recovered and fluorescence as well asproductivity were determined. The values of fluorescence level andproductivity are shown in the brief description of FIG. 14.

3.5. Cloning of sorted pools by the FACS ACDU device—isolation ofclones. Following three consecutive sorting cycles, The top ˜4%fluorescent cells of anti-IL22RA mAb producing pools #2418UN-Hx3 inProCHO5 were cloned by means of the Automated Cell Deposition Unit(ACDU) device of the FACSAria, which enables single cell cloning into 96well plates. Cloning of cells grown in ProCHO5+20 mg/ml puromycin and 25μM/m MSX was done in 96 well plates containing 80% medium C6366 and 20%C6614. Colonies were detected by Cellavista at seeding day and thenevery 2-3 days. Wells with more than one colony were discarded. Fromcloning onward the culture media did not contain puromycin.

Supernatants from 358 wells were sampled and assayed for productivity(titer) by ELISA (FIG. 15). Forty clones derived from the pool with thehighest titers were transferred first to T25 flasks and then to 50 mltubes for evaluating specific productivity (FIG. 16). The best 15 clonesderived from both pools were cultured with 25 μM MSX for evaluation ofproductivity in 50 ml tubes (FIG. 17).

3.6 Protein product identification in crude cell culture medium by SDSPAGE Western Blot. The candidate clones were grown in ProCHO-5 medium.Product produced by those clones in the crude cell culture medium wasanalyzed (FIG. 18) by SDS-PAGE/Western blotting. Detection of theproducts was done with antibodies to human Fc and to the kappa lightchain subunits of anti-IL22RA mAb (A and B respectively).

The results show that the intact product (heterodimer), with theapparent molecular weight of ˜150 kDa was identified by both antibodies.In addition, free light chain secreted by all candidate clones (monomerand dimer according to apparent MW) was observed on the Western blotsstained with the anti-LC (FIGS. 18B and 19B).

Molecular weight of the heterodimer in the crude cell culture mediumfrom all clones conforms to that of the purified product from theanti-IL22RA mAb reference sample. No significant difference in theapparent MW of the products of the different clones was found. Multiplebands may represent glycosylation micro-heterogeneity.

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1. A nucleic acid molecule comprising: (a) a first nucleic acid sequenceencoding a signal peptide, linked at its C-terminal to (b) a secondnucleic acid sequence encoding a biotin mimetic peptide (BMP) or abiotin acceptor peptide (BAP), linked at its C-terminal to (c) a thirdnucleic acid sequence encoding a polypeptide stretch that allows theanchorage of the BMP or BAP to a cell membrane.
 2. The nucleic acidmolecule according to claim 1, wherein the first and second nucleic acidsequences) and/or the second and third nucleic acid sequences are linkedby a nucleic acid sequence encoding a linker peptide.
 3. The nucleicacid molecule according to claim 1, wherein the polypeptide stretch thatallows the anchorage of the BMP or BAP to a cell membrane is CD59a ofSEQ ID NO:
 9. 4. The nucleic acid molecule according to claim 1, whereinthe polypeptide stretch that allows the anchorage of the BMP or BAP to acell membrane is the synthetic carrier peptide of the amino acidsequence of SEQ ID NO: 1 directly linked to CD48-GPI.
 5. The nucleicacid molecule according to claim 1, wherein the polypeptide stretch thatallows the anchorage of the BMP or BAP to a cell membrane is thesynthetic carrier peptide of the SEQ ID NO: 1 linked to a transmembranepeptide.
 6. The nucleic acid molecule according to claim 5, wherein thetransmembrane peptide is IGF-1R-TM of ID SEQ NO:
 2. 7. The nucleic acidmolecule according to claim 1, wherein the signal peptide is the signalpeptide of human growth hormone or of CD59.
 8. The nucleic acid moleculeaccording to claim 1, wherein the polypeptide stretch that allows theanchorage of the BMP or BAP to a cell membrane is linked at itsC-terminal to a polypeptide conferring selection resistance.
 9. Thenucleic acid molecule according to claim 8, wherein the polypeptideconferring selection resistance is selected from the group consisting ofan antibiotic-hydrolyzing enzyme, an antibiotic-scavenging protein, ametabolic enzyme and an antibiotic modifying enzyme.
 10. The nucleicacid molecule according to claim 8, wherein the polypeptide stretch thatallows the anchorage of the BMP or BAP to a cell membrane is linked topolypeptide conferring-selection resistance via a linker peptide. 11.The nucleic acid molecule according to claim 9, wherein theantibiotic-hydrolyzing enzyme is selected from the group consisting of apuromycin-hydrolyzing enzyme, such as puromycin N-acetyl transferase(PAC) and an aminoglycoside hydrolyzing enzyme, such as aminoglycoside3′-phosphotransferase; the antibiotic-scavenging protein is an Sh blegene product; and the antibiotics modifying enzyme is a hygromycinkinase.
 12. The nucleic acid molecule according to claim 2, wherein thelinker peptide consists of the amino acid sequence of isoleucine andproline or it consists of an amino acid sequence of SEQ ID NO:
 4. 13.The nucleic acid molecule according to claim 1, which is operably linkedto a promoter capable of driving the transcription of the nucleic acidmolecule, and is optionally operably linked upstream to an EMCV IRESsequence.
 14. The nucleic acid molecule according to claim 13, whereinthe promoter is hCMV.
 15. The nucleic acid molecule according to claim1, wherein the second nucleic acid sequence codes for a BMP.
 16. Thenucleic acid molecule according to claim 15, wherein the BMP comprisesthe amino acid sequence of SEQ ID NO: 5
 17. The nucleic acid moleculeaccording to claim 15, wherein the nucleic acid molecule furthercomprises nucleic acid sequences encoding the synthetic carrier peptideof the amino acid sequence of SEQ ID NO: 1 directly linked to thetransmembrane peptide of the amino acid sequence of SEQ ID NO: 2, thesignal peptide of CD59a of the amino acid sequence of SEQ ID NO: 6, andPAC of the amino acid sequence of SEQ ID NO:
 7. 18. The nucleic acidmolecule according to claim 17, comprising the nucleic acid sequence ofSEQ ID NO:
 8. 19. The nucleic acid molecule according to claim 15,wherein the nucleic acid molecule further comprises nucleic acidsequences encoding mouse CD59a of the amino acid sequence of SEQ ID NO:9, and the signal peptide of CD59a of the amino acid sequence of SEQ IDNO:
 6. 20. The nucleic acid molecule according to claim 18, wherein thenucleic acid sequence is of SEQ ID NO:
 10. 21. The nucleic acid moleculeaccording to claim 14, wherein the nucleic acid molecule furthercomprises nucleic acid sequences encoding the synthetic carrier peptideof the amino acid sequence of SEQ ID NO: 1 directly linked to thetransmembrane peptide of the amino acid sequence of SEQ ID NO: 2, andthe signal peptide of CD59a of the amino acid sequence of SEQ ID NO: 6.22. The nucleic acid molecule according to claim 20, wherein the nucleicacid sequence is of SEQ ID NO:
 11. 23. The nucleic acid moleculeaccording to claim 14, wherein the nucleic acid molecule furthercomprises nucleic acid sequences encoding the synthetic carrier peptideof the amino acid sequence of SEQ ID NO: 1 directly linked to theCD48-GPI anchor peptide of the amino acid sequence of SEQ ID NO: 3, andthe signal peptide of CD59a of the amino acid sequence of SEQ ID NO: 6.24. The nucleic acid molecule according to claim 22, wherein the nucleicacid sequence is of SEQ ID NO:
 12. 25. The nucleic acid moleculeaccording to claim 1, further comprising: a POI-encoding nucleic acidsequence.
 26. The nucleic acid molecule according to claim 25, whereinthe POI-encoding nucleic acid sequence is operably linked upstream to anEMCV IRES sequence or downstream to a promoter capable of driving thetranscription of the nucleic acid molecule, or both.
 27. The nucleicacid molecule according to claim 25, wherein the POI-encoding nucleicacid sequence is a POI-encoding nucleic acid sequence coding for asingle polypeptide.
 28. The nucleic acid molecule according to claim 25,wherein the POI-encoding nucleic acid sequence comprises twoPOI-encoding nucleic acid sequences coding for different subunits of aPOI.
 29. The nucleic acid molecule according to claim 28, wherein thesubunits are the light and heavy chains of an antibody.
 30. The nucleicacid molecule according to claim 25, wherein the protein of interest islinked at its amino terminus, optionally via a linker peptide, to asignal peptide. 31.-42. (canceled)
 43. The nucleic acid moleculeaccording to claim 26, wherein the POI is selected from the groupconsisting of an Fc-fusion product, an antibody, a cytokine, a hormone,a growth factor, a neurotransmitter, an enzyme, a receptor ligand, asialomucin, a nuclear protein, a regulatory protein and a toxin, or afunctional fraction thereof.
 44. A vector comprising the nucleic acidmolecule according to claim
 1. 45. A method for the selection ofeukaryotic cells secreting a protein of interest (POI), comprisingidentifying cells presenting BMP on their cell surface, wherein thelevel of BMP presentation on the cell surface is correlated with theamount of POI secreted, the method comprising the steps of: (d)transfecting cells with a vector according to claim 44, in which thesecond nucleic acid sequence codes for BMP and a second vectorcomprising aPOI-encoding nucleic acid sequence; or with a vectorcomprising the nucleic acid molecule further comprising a POI-encodingnucleic acid sequence, in which the second nucleic acid sequence codesfor BMP, thereby establishing a stable pool of BMP transfected cells;(e) labeling the BMP transfected cells with a detectable biotin-bindingmoiety; and (f) identifying and isolating transfected cells labeled withthe detectable biotin-binding moiety.
 46. A method for the selection ofeukaryotic cells secreting a protein of interest (POI), comprisingidentifying cells presenting biotin on their cell surface, wherein thelevel of biotin presentation on the cell surface is correlated with theamount of POI secreted, the method comprising the steps of: (a)transfecting cells with a vector according to claim 32 wherein thesecond nucleic acid sequence codes for BAP and a second vectorcomprising a POI-encoding nucleic acid sequence; or a vector comprisingthe nucleic acid molecule further comprising a POI-encoding nucleic acidsequence, wherein the second nucleic acid sequence codes for BAP,thereby establishing a stable pool of BAP transfected cells; (b) addingBirA and biotin, (c) labeling the BAP transfected cells with abiotin-binding moiety; and (d) identifying and isolating transfectedcells labeled with the detectable biotin-binding moiety.
 47. The methodaccording to claim 46, further comprising propagating the isolated cellsand repeating steps (b) to (d).
 48. The method according to claim 47,further comprising isolating a single cell labeled with the detectablebiotin binding moiety and propagating the cell to form a clone.
 49. Themethod according to claim 48, further comprising detecting andquantifying the secreted protein of interest, thereby selectingeukaryotic cells secreting a protein of interest.
 50. The methodaccording to claims 46, wherein said cells present BMP on their cellsurface.
 51. The method according to claim 46, wherein said cells areeukaryotic cells selected from mammalian, plant, insect and yeast cells.52. The method according to claim 51, wherein the mammalian cells areselected from Chinese Hamster Ovary (CHO) cells, baby mouse myeloma NS0cells, hamster kidney (BHK) cells, human embryo kidney (HEK) cells,human retinal cells, COS cells, SP2/0 cells, WI38 cells, MRCS cells andPer.C6 cells.
 53. The method according to claim 46, wherein the cellsare labeled by contacting them with a detectable biotin-binding moietyselected from the group consisting of fluorescent avidin and fluorescentstreptavidin.
 54. The method according to claim 46, wherein the labeledcells are identified and isolated by the means of a FACS or magnetbeads.
 55. The method according to claim 53, wherein the selectedeukaryotic cells are presenting higher amounts of BMP on their surfaceand are secreting larger amounts of protein of interest than thetransfected cells of the stable pool.
 56. The method according to claim55, wherein the amount of BMP on the surface of the selected eukaryoticcells and the amount of protein of interest secreted by the selectedeukaryotic cells are larger by at least a factor selected from the groupconsisting of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 and up to30-fold higher than the transfected cells of the stable pool.